The present invention relates to the field of labelling reagents. In particular, the invention relates to cyanine dyes having meso-substituents in the polymethine chain linking the heterocyclic systems. The invention also relates to assay methods utilising such dyes.
There is increasing interest in, and demand for, fluorescent reporters for use in the labelling and detection of biomolecules. Cyanine and related dyes offer a number of advantages over other fluorescent dye reagents and they are finding widespread use as fluorescent labels in such diverse areas as sequencing, microarrays, flow cytometry and proteomics. The cyanine dyes are characterised by having very high extinction coefficients and favourable quantum yields. In addition, cyanine dyes possess good photostability and are not photobleached. The dyes have been used extensively as labels in biological studies.
U.S. Pat. No. 6,048,982 (Waggoner, A. S.) discloses luminescent cyanine dyes having the structure:
wherein X and Y are independently selected from the group consisting of O, S and CH3—C—CH3; m is an integer from 1–4 and at least one of the groups R1, R2, R3, R4 and R7 is a reactive group, reactive with amino, sulphydryl or hydroxy nucleophiles.
WO 02/26891 (Molecular Probes Inc.) discloses cyanine dyes incorporating similar structures.
In cyanine dyes of the above formula, the number of methine groups linking the heterocyclic ring systems defines the absorption maxima of the dyes. Thus, the absorption maxima increase from Cy™3 to Cy5 to Cy7 by an increment of approximately 100 nm each as illustrated below. The corresponding emission peaks of Cy3, Cy5 and Cy7 are also separated by approximately 100 nm, as shown in Table 1.
TABLE 1StructureAbsEmCompoundX = Y = C(CH3)2max (nm)max (nm)Cy3n = 1550570Cy5n = 2649670Cy7n = 3743767
Moreover, the absorption and emission maxima are not much affected by the introduction of either electron withdrawing or donating substitution(s) on the phenyl rings of the heterocycles. Substitution of the phenyl rings with naphthyl rings has been shown to offer the largest bathochromic shifts for the indocyanine dyes giving rise to Cy3.5 and Cy5.5 dyes having spectral characteristics as shown in Table 2.
TABLE 2StructureAbsEmCompoundX = Y = C(CH3)2max (nm)max (nm)Cy3.5n = 1581596Cy5.5n = 2675694
In many applications that utilise fluorescent dyes, especially for high throughput DNA sequencing, it is desirable to have highly fluorescent, functionalised dyes with absorption maxima (and therefore, emission maxima) having approximately 25 to 30 nm spacing in the entire spectral range from 550 nm to 750 nm. Thus, there is requirement for additional dyes having spectral characteristics that complement those listed above. British Patent No. 620801 (Ilford Ltd) relates to the preparation of cyanine dyestuffs having a meso-substituted cyano group. The spectral properties of the dyes are not disclosed however. Chibisov, A. K. et al (J.Chem.Soc. Faraday Trans., (1996), 92(24), 4917–25) describe a spectroscopic study involving indocarbocyanines with substituents in the meso position of the polymethine chain. These dyes have not been described for use in the labelling and detection of materials, particularly biological molecules.